Disruption of Circadian Clock Induces Abnormal Mammary Morphology and Aggressive Basal Tumorigenesis by Enhancing LILRB4 Signaling.

Epidemiological studies have shown that circadian rhythm disruption (CRD) is associated with the risk of breast cancer. However, the role of CRD in mammary gland morphology and aggressive basal mammary tumorigenesis and the molecular mechanisms underlying CRD and cancer risk remain unknown. To investigate the effect of CRD on aggressive tumorigenesis, a genetically engineered mouse model that recapitulates the human basal type of breast cancer was used for this study. The effect of CRD on mammary gland morphology was investigated using wild-type mice model. The impact of CRD on the tumor microenvironment was investigated using the tumors from LD12:12 and CRD mice via scRNA seq. ScRNA seq was substantiated by multiplexing immunostaining, flow cytometry, and realtime PCR. The effect of LILRB4 immunotherapy on CRD-induced tumorigenesis was also investigated. Here we identified the impact of CRD on basal tumorigenesis and mammary gland morphology and identified the role of LILRB4 on CRD-induced lung metastasis. We found that chronic CRD disrupted mouse mammary gland morphology and increased tumor burden, and lung metastasis and induced an immunosuppressive tumor microenvironment by enhancing LILRB4a expression. Moreover, CRD increased the M2-macrophage and regulatory T-cell populations but decreased the M1-macrophage populations. Furthermore, targeted immunotherapy against LILRB4 reduced CRD-induced immunosuppressive microenvironment and lung metastasis. These findings identify and implicate LILRB4a as a link between CRD and aggressive mammary tumorigenesis. This study also establishes the potential role of the targeted LILRB4a immunotherapy as an inhibitor of CRD-induced lung metastasis.

Influence of Housing Temperature and Genetic Diversity on Allogeneic T Cell-Induced Tissue Damage in Mice.

The objective of this study was to determine how housing temperature and genetic diversity affect the onset and severity of allogeneic T cell-induced tissue damage in mice subjected to reduced intensity conditioning (RIC). We found that adoptive transfer of allogeneic CD4+ T cells from inbred donors into sub-lethally irradiated inbred recipients (I→I) housed at standard housing temperatures (ST; 22-24 °C) induced extensive BM and spleen damage in the absence of injury to any other tissue. Although engraftment of T cells in RIC-treated mice housed at their thermo-neutral temperature (TNT; 30-32 °C) also developed similar BM and spleen damage, their survival was markedly and significantly increased when compared to their ST counterparts. In contrast, the adoptive transfer of allogeneic T cells into RIC-treated outbred CD1 recipients failed to induce disease in any tissue at ST or TNT. The lack of tissue damage was not due to defects in donor T cell trafficking to BM or spleen but was associated with the presence of large numbers of B cells and myeloid cells within these tissues that are known to contain immunosuppressive regulatory B cells and myeloid-derived suppressor cells. These data demonstrate, for the first time, that housing temperature affects the survival of RIC-treated I→I mice and that RIC-conditioned outbred mice are resistant to allogeneic T cell-induced BM and spleen damage.

Ixodes scapularis nymph saliva protein blocks host inflammation and complement-mediated killing of Lyme disease agent, Borrelia burgdorferi.

Tick serine protease inhibitors (serpins) play crucial roles in tick feeding and pathogen transmission. We demonstrate that Ixodes scapularis (Ixs) nymph tick saliva serpin (S) 41 (IxsS41), secreted by Borrelia burgdorferi (Bb)-infected ticks at high abundance, is involved in regulating tick evasion of host innate immunity and promoting host colonization by Bb. Recombinant (r) proteins were expressed in Pichia pastoris, and substrate hydrolysis assays were used to determine. Ex vivo (complement and hemostasis function related) and in vivo (paw edema and effect on Bb colonization of C3H/HeN mice organs) assays were conducted to validate function. We demonstrate that rIxsS41 inhibits chymase and cathepsin G, pro-inflammatory proteases that are released by mast cells and neutrophils, the first immune cells at the tick feeding site. Importantly, stoichiometry of inhibition analysis revealed that 2.2 and 2.8 molecules of rIxsS41 are needed to 100% inhibit 1 molecule of chymase and cathepsin G, respectively, suggesting that findings here are likely events at the tick feeding site. Furthermore, chymase-mediated paw edema, induced by the mast cell degranulator, compound 48/80 (C48/80), was blocked by rIxsS41. Likewise, rIxsS41 reduced membrane attack complex (MAC) deposition via the alternative and lectin complement activation pathways and dose-dependently protected Bb from complement killing. Additionally, co-inoculating C3H/HeN mice with Bb together with rIxsS41 or with a mixture (rIxsS41 and C48/80). Findings in this study suggest that IxsS41 markedly contributes to tick feeding and host colonization by Bb. Therefore, we conclude that IxsS41 is a potential candidate for an anti-tick vaccine to prevent transmission of the Lyme disease agent.

Hypoxia-inducible-factors differentially contribute to clinical disease and the control of viral replication during RSV infection.

Hypoxia-inducible-factors (HIF) are transcription factors that regulate cellular adaptation to hypoxic conditions, enabling cells to survive in low-oxygen environments. Viruses have evolved to stabilize this pathway to promote successful viral infection, therefore modulation of HIFs could represent a novel antiviral strategy. In previous in vitro studies, we found that respiratory syncytial virus (RSV), a leading cause of respiratory illness, stabilizes HIFs under normoxic conditions, with inhibition of HIF-1α resulting in reduced viral replication. Despite several HIF modulating compounds being tested/approved for use in other non-infectious models, little is known about their efficacy against respiratory viruses using relevant animal models. This study aimed to characterize the disease modulating properties and antiviral potential of anti-HIF-1α (PX478) and anti-HIF-2α (PT2385) in RSV-infected BALB/c mice. We found that inhibition of HIF-1α worsen clinical disease parameters, while simultaneously improving airway function. Additionally, anti-HIF-1α results in significantly reduced viral titer at early and peak time points of RSV replication, followed by a loss in viral clearance when given every day, but not every-other-day. In contrast, inhibition of HIF-2α was associated with improved clinical parameters, with no changes in airway function, and amelioration of interstitial pneumonia. Furthermore, anti-HIF-2α reduced early and peak lung viral replication, with no impairment of viral clearance. Analysis of lung cells found significant modification in the T cell compartment that correlated with changes in lung pathology and viral titers in response to each HIF inhibitor administration. These data underscore the complex role of HIFs in RSV infection and highlight the need for careful therapeutic consideration.

Micro-CT Features of Lung Consolidation, Collagen Deposition and Inflammation in Experimental RSV Infection Are Aggravated in the Absence of Nrf2.

Severe respiratory syncytial virus (RSV) infections in early life have been linked to the development of chronic airway disease. RSV triggers the production of reactive oxygen species (ROS), which contributes to inflammation and enhanced clinical disease. NF-E2-related factor 2 (Nrf2) is an important redox-responsive protein that helps to protect cells and whole organisms from oxidative stress and injury. The role of Nrf2 in the context of viral-mediated chronic lung injury is not known. Herein, we show that RSV experimental infection of adult Nrf2-deficient BALB/c mice (Nrf2-/-; Nrf2 KO) is characterized by enhanced disease, increased inflammatory cell recruitment to the bronchoalveolar compartment and a more robust upregulation of innate and inflammatory genes and proteins, compared to wild-type Nrf2+/+ competent mice (WT). These events that occur at very early time points lead to increased peak RSV replication in Nrf2 KO compared to WT mice (day 5). To evaluate longitudinal changes in the lung architecture, mice were scanned weekly via high-resolution micro-computed tomography (micro-CT) imaging up to 28 days after initial viral inoculation. Based on micro-CT qualitative 2D imaging and quantitative reconstructed histogram-based analysis of lung volume and density, we found that RSV-infected Nrf2 KO mice developed significantly greater and prolonged fibrosis compared to WT mice. The results of this study underscore the critical role of Nrf2-mediated protection from oxidative injury, not only in the acute pathogenesis of RSV infection but also in the long-term consequences of chronic airway injury.

SARS-CoV-2 Inhibits NRF2-Mediated Antioxidant Responses in Airway Epithelial Cells and in the Lung of a Murine Model of Infection.

Several viruses have been shown to modulate the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of redox homeostasis. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, also seems to disrupt the balance between oxidants and antioxidants, which likely contributes to lung damage. Using in vitro and in vivo models of infection, we investigated how SARS-CoV-2 modulates the transcription factor NRF2 and its dependent genes, as well as the role of NRF2 during SARS-CoV-2 infection. We found that SARS-CoV-2 infection downregulates NRF2 protein levels and NRF2-dependent gene expression in human airway epithelial cells and in lungs of BALB/c mice. Reductions in cellular levels of NRF2 seem to be independent of proteasomal degradation and the interferon/promyelocytic leukemia (IFN/PML) pathway. Furthermore, lack of the Nrf2 gene in SARS-CoV-2-infected mice exacerbates clinical disease, increases lung inflammation, and is associated with a trend toward increased lung viral titers, indicating that NRF2 has a protective role during this viral infection. In summary, our results suggest that SARS-CoV-2 infection alters the cellular redox balance by downregulating NRF2 and its dependent genes, which exacerbates lung inflammation and disease, therefore, suggesting that the activation of NRF2 could be explored as therapeutic approach during SARS-CoV-2 infection. IMPORTANCE The antioxidant defense system plays a major function in protecting the organism against oxidative damage caused by free radicals. COVID-19 patients often present with biochemical characteristics of uncontrolled pro-oxidative responses in the respiratory tract. We show herein that SARS-CoV-2 variants, including Omicron, are potent inhibitors of cellular and lung nuclear factor erythroid 2-related factor 2 (NRF2), the master transcription factor that controls the expression of antioxidant and cytoprotective enzymes. Moreover, we show that mice lacking the Nrf2 gene show increased clinical signs of disease and lung pathology when infected with a mouse-adapted strain of SARS-CoV-2. Overall, this study provides a mechanistic explanation for the observed unbalanced pro-oxidative response in SARS-CoV-2 infections and suggests that therapeutic strategies for COVID-19 may consider the use of pharmacologic agents that are known to boost the expression levels of cellular NRF2.

Combining gamma-tocopherol and aspirin synergistically suppresses colitis-associated colon tumorigenesis and modulates the gut microbiota in mice, and inhibits the growth of human colon cancer cells.

Despite being shown to be effective for chemoprevention of colorectal cancer, aspirin has limitations including adverse effects and inability to block colitis-associated colon cancer (CAC). γ-Tocopherol (γT), a vitamin E form, has been reported to mitigate experimental colitis and CAC, prolong the anti-inflammatory activity of aspirin and alleviate aspirin-induced adverse effect. We therefore hypothesize that combining γT and aspirin is better than either compound singly for suppressing CAC. This hypothesis was tested in the murine azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CAC model and with human HCT116 colon cancer cells. Compared to the control, combining aspirin (250 ppm) and γT (500 ppm) but not either compound alone significantly reduced AOM/DSS-induced tumor area and multiplicity of large-size tumors by 60% and 50%, respectively. Meanwhile, γT mitigated aspirin-promoted inflammation and stomach lesions in mice. Moreover, the combination appeared to cause favorable changes of gut microbiota compared to the control and synergistically suppressed the growth of HCT116 cells. Our study demonstrates that combining aspirin and γT improves anticancer effects and counteracts side effects compared to aspirin and may therefore be a novel combinatory chemopreventive agent against CAC.

Viral Clearance and Neuroinflammation in Acute TMEV Infection Vary by Host Genetic Background.

A wide range of viruses cause neurological manifestations in their hosts. Infection by neurotropic viruses as well as the resulting immune response can irreversibly disrupt the complex structural and functional architecture of the brain, depending in part on host genetic background. The interaction between host genetic background, neurological response to viral infection, and subsequent clinical manifestations remains poorly understood. In the present study, we used the genetically diverse Collaborative Cross (CC) mouse resource to better understand how differences in genetic background drive clinical signs and neuropathological manifestations of acute Theiler's murine encephalomyelitis virus (TMEV) infection. For the first time, we characterized variations of TMEV viral tropism and load based on host genetic background, and correlated viral load with microglial/macrophage activation. For five CC strains (CC002, CC023, CC027, CC057, and CC078) infected with TMEV, we compared clinical signs, lesion distribution, microglial/macrophage response, expression, and distribution of TMEV mRNA, and identified genetic loci relevant to the early acute (4 days post-infection [dpi]) and late acute (14 dpi) timepoints. We examined brain pathology to determine possible causes of strain-specific differences in clinical signs, and found that fields CA1 and CA2 of the hippocampal formation were especially targeted by TMEV across all strains. Using Iba-1 immunolabeling, we identified and characterized strain- and timepoint-specific variation in microglial/macrophage reactivity in the hippocampal formation. Because viral clearance can influence disease outcome, we used RNA in situ hybridization to quantify viral load and TMEV mRNA distribution at both timepoints. TMEV mRNA expression was broadly distributed in the hippocampal formation at 4 dpi in all strains but varied between radiating and clustered distribution depending on the CC strain. We found a positive correlation between microglial/macrophage reactivity and TMEV mRNA expression at 4 dpi. At 14 dpi, we observed a dramatic reduction in TMEV mRNA expression, and localization to the medial portion of field CA1 and field CA2. To better understand how host genetic background can influence pathological outcomes, we identified quantitative trait loci associated with frequency of lesions in a particular brain region and with microglial/macrophage reactivity. These QTL were located near several loci of interest: lysosomal trafficking regulator (Lyst) and nidogen 1 (Nid1), and transmembrane protein 106 B (Tmem106b). Together, these results provide a novel understanding about the influences of genetic variation on the acute neuropathological and immunopathological environment and viral load, which collectively lead to variable disease outcomes. Our findings reveal possible avenues for future investigation which may lead to more effective intervention strategies and treatment regimens.

Nested PCR Detection of Pythium sp. from Formalin-Fixed, Paraffin-Embedded Canine Tissue Sections.

Pythium insidiosum is an infectious oomycete affecting dogs that develop the cutaneous or gastrointestinal form of pythiosis with a poor prognosis. If left untreated, pythiosis may be fatal. This organism is not a true fungus because its cell wall and cell membrane lack chitin and ergosterol, respectively, requiring specific treatment. Identifying the organism is challenging, as a hematoxylin and eosin (H&E) stain poorly stain the P. insidiosum hyphae and cannot be differentiated conclusively from other fungal or fungal-like organisms (such as Lagenidium sp.) morphologically. Our study aimed to develop a nested PCR to detect P. insidiosum and compare it with the traditional histopathologic detection of hyphae. Formalin-fixed, paraffin-embedded (FFPE) tissue scrolls from 26 dogs with lesions suggesting the P. insidiosum infection were assessed histologically, and DNA was extracted from the FFPE tissue sections for nested PCR. Agreement between the histologic stains, (H&E), periodic acid-Schiff (PAS), and/or Grocott methenamine silver (GMS) and the nested PCR occurred in 18/26 cases. Hyphae consistent with Pythium sp. were identified via histopathology in 57.7% of the samples, whereas the nested PCR detected P. insidiosum in 76.9% of samples, aiding in the sensitivity of the diagnosis of pythiosis in dogs. Using this combination of techniques, we report 20 canine cases of pythiosis over 18 years in Indiana and Kentucky, an unexpectedly high incidence for temperate climatic regions. Using a combination of histopathology evaluation and nested PCR is recommended to aid in the accurate diagnosis of pythiosis.

Supplementation of polyphenol-rich grapes attenuates colitis, colitis-associated colon cancer, and disease-associated dysbiosis in mice, but fails to mitigate colitis in antibiotic-treated mice.

Polyphenols are known to interact with gut microbes that play key roles in maintaining gut health, but the role of gut microbiota modulation by polyphenols in mitigating colonic diseases is not fully established. We hypothesize that the interaction of polyphenols with the gut microbiota contributes to the attenuation of colitis and colitis-associated colon cancer (CAC). To test this hypothesis, we examined the effects of dietary supplementation of polyphenol-rich grape powder (GP) on azoxymethane (AOM) and dextran sulfate sodium (DSS)-induced colitis, CAC, and the gut microbiota in mice (study 1), and further compared anti-colitis effects of GP in regular and antibiotic-treated mice (study 2). Compared to the control diet that has matched non-polyphenol contents, 10% GP, but not 3% GP, attenuated AOM-DSS-induced colitis and tumor multiplicity by 29% (P<.05). Ten percent GP increased gut bacterial evenness and counteracted CAC-induced decrease of bacterial evenness and changes in microbial composition. Remarkably, the estimated gut bacterial functional profiles of healthy mice and diseased mice fed 10% GP were similar, and both were significantly different from those of diseased mice fed the control diet. Furthermore, 10% GP increased the relative abundance of butyrate-producing bacteria in the Lachnospiraceae family and enhanced the concentrations of fecal butyrate. Additionally, 10% GP mitigated DSS-induced colitis in conventional mice, but not the antibiotic-treated, gut microbe-depleted mice. Collectively, our studies demonstrate that grape polyphenols alleviate colonic diseases and prevent disease-associated dysbiosis, and their interaction with the gut microbiota may play a causative role in the protection of gut health.

An engineered dual function peptide to repair fractured bones.

Targeted drug delivery, often referred to as "smart" drug delivery, is a process whereby a therapeutic drug is delivered to specific parts of the body in a manner that increases its concentration at the desired sites relative to others. This approach is poised to revolutionize medicine as exemplified by the recent FDA approval of Cytalux (FDA approves pioneering drug for ovarian cancer surgery - Purdue University News) which is a folate-receptor targeted intraoperative near infrared (NIR) imaging agent that was developed in our laboratories. Fracture-associated morbidities and mortality affect a significant portion of world population. United states, Canada and Europe alone spent $48 billion in treating osteoporosis related fractures although this number doesn't count the economic burden due to loss in productivity. It is estimated that by 2050 ca 21 million hip fractures would occur globally which will be leading cause of premature death and disability. Despite the need for improvement in the treatment for fracture repair, methods for treating fractures have changed little in recent decades. Systemic delivery of fracture-homing bone anabolics holds great promise as a therapeutic strategy in this regard. Here we report the design of a fracture-targeted peptide comprised of a payload that binds and activates the parathyroid hormone receptor (PTHR1) and is linked to a targeting ligand comprised of 20 D-glutamic acids (D-Glu20) that directs accumulation of the payload specifically at fracture sites. This targeted delivery results in reduction of fracture healing times to <1/2 while creating repaired bones that are >2-fold stronger than saline-treated controls in mice. Moreover, this hydroxyapatite-targeted peptide can be administered without detectable toxicity to healthy tissues or modification of healthy bones in dogs. Additionally, since similar results are obtained upon treatment of osteoporotic and diabetic fractures in mice, and pain resolution is simultaneously accelerated by this approach, we conclude that this fracture-targeted anabolic peptide displays significant potential to revolutionize the treatment of bone fractures.

Implementing Digital Pathology into Veterinary Academics and Research.

The advantages of digital pathology (DP) have been recognized as early as 1963, but only within the last decade or so have the advancements of slide scanners and viewing software made the use and implementation of DP feasible in the classroom and in research. Several factors must be considered prior to undertaking the project of implementing the DP workflow in any setting, but particularly in an academic environment. Sustained and open dialogue with information technology (IT) is critical to the success of this enterprise. In addition to IT, there is a multitude of criteria to consider when determining the best hardware and software to purchase to support the project. The goals and limitations of the laboratory and the requirements of its users (students, instructors, and researchers) will ultimately direct these decisions. The objectives of this article are to provide an overview of the opportunities and challenges associated with the integration of DP in education and research, to highlight some important IT considerations, and to discuss some of the requirements and functionalities of some hardware and software options.

Prevalence of Neospora caninum Exposure in Wild Pigs (Sus scrofa) from Oklahoma with Implications of Testing Method on Detection.

Neospora caninum is a protozoan parasite, reported as a leading cause of cattle abortions and reproductive failure worldwide, costing the cattle industry approximately $1.3 billion annually. With wild pig (Sus scrofa) populations estimated at over six million in the United States, contact between wild pigs and livestock is inevitable, mainly because of the widespread geographic co-occurrence of the two species. As a known reservoir for numerous fungal, bacterial, viral and parasitic diseases, wild pigs are of particular importance for human and veterinary health relative to the prevention of infectious diseases. The seroprevalence of N. caninum in wild pig populations was previously documented in the United States, raising the question as to their exposure point of prevalence. This research screened 116 individual wild pigs for N. caninum using a variety of available assays. Using two different commercially available ELISA test kits, seroprevalence ranged from 12.5% to 67.8%. The Indirect Fluorescent Antibody Test resulted in our highest percent seroprevalence for these samples, at 84.1%. However, none of our samples showed any presence of N. caninum or associated pathologies via histological evaluation of representative tissues. Importantly, the assays used in this study were not congruent with all duplicate samples or between the test types used. The implications of these non-congruent results demonstrates that currently available testing assays produce variable results, underscoring the need for more reliable testing kits and a standardized methodology when assessing disease prevalence in wildlife, particularly for N. caninum in wild pigs, which impacts prevalence and comparability across studies.

Antibiotic administration exacerbates acute graft vs. host disease-induced bone marrow and spleen damage in lymphopenic mice.

BACKGROUND: Hematopoietic stem cell transplantation is a potential cure for certain life-threatening malignant and nonmalignant diseases. However, experimental and clinical studies have demonstrated that pre-transplant myeloablative conditioning damages the gut leading to translocation of intestinal bacteria and the development of acute graft vs. host disease (aGVHD). The overall objective of this study was to determine whether administration of broad spectrum antibiotics (Abx) affects the onset and/or severity of aGVHD in lymphopenic mice that were not subjected to toxic, pre-transplant conditioning. RESULTS: We found that treatment of NK cell-depleted recombination activating gene-1-deficient (-NK/RAG) recipients with an Abx cocktail containing vancomycin and neomycin for 7 days prior to and 4 weeks following adoptive transfer of allogeneic CD4+ T cells, exacerbated the development of aGVHD-induced BM failure and spleen damage when compared to untreated-NK/RAG recipients engrafted with syngeneic or allogeneic T cells. Abx-treated mice exhibited severe anemia and monocytopenia as well as marked reductions in BM- and spleen-residing immune cells. Blinded histopathological analysis confirmed that Abx-treated mice engrafted with allogeneic T cells suffered significantly more damage to the BM and spleen than did untreated mice engrafted with allogeneic T cells. Abx-induced exacerbation of BM and spleen damage correlated with a dramatic reduction in fecal bacterial diversity, marked loss of anaerobic bacteria and remarkable expansion of potentially pathogenic bacteria. CONCLUSIONS: We conclude that continuous Abx treatment may aggravate aGVHD-induced tissue damage by reducing short chain fatty acid-producing anaerobes (e.g. Clostridium, Blautia) and/or by promoting the expansion of pathobionts (e.g. Akkermansia) and opportunistic pathogens (Cronobacter).

A zero-inflated non-negative matrix factorization for the deconvolution of mixed signals of biological data.

A latent factor model for count data is popularly applied in deconvoluting mixed signals in biological data as exemplified by sequencing data for transcriptome or microbiome studies. Due to the availability of pure samples such as single-cell transcriptome data, the accuracy of the estimates could be much improved. However, the advantage quickly disappears in the presence of excessive zeros. To correctly account for this phenomenon in both mixed and pure samples, we propose a zero-inflated non-negative matrix factorization and derive an effective multiplicative parameter updating rule. In simulation studies, our method yielded the smallest bias. We applied our approach to brain gene expression as well as fecal microbiome datasets, illustrating the superior performance of the approach. Our method is implemented as a publicly available R-package, iNMF.

Feasibility of a mini-pig model of radiation-induced brain injury to one cerebral hemisphere.

BACKGROUND: Radiation-induced brain injury is a common concern for survivors of adult and pediatric brain cancer. Pre-clinically, rodent models are the standard approach to evaluate mechanisms of injury and test new therapeutics for this condition. However, these rodent models fail to recapitulate the radiological and histological characteristics of the clinical disease. METHODS: Here we describe a hemispheric mini-pig model of radiation-induced brain injury generated with a clinical 6 MV photon irradiator and evaluated with a clinical 3T MRI. Two pairs of Yucatan mini-pigs each received either 15 Gy or 25 Gy to the left brain hemisphere. Quality of intensity modulated radiation therapy treatment plans was evaluated retrospectively with parameters reported according to ICRU guidelines. The pigs were observed weekly to check for any outright signs of neurological impairment. The pigs underwent anatomical MRI examination before irradiation and up to 6 months post-irradiation. Immediately after the last imaging time point, the pigs were euthanized and their brains were collected for histopathological assessment. RESULTS: Analysis of the dose volume histograms showed that 93% of the prescribed dose was delivered to at least 93% of the target volume in the left hemisphere. Organs at risk excluded from the target volume received doses below clinical safety thresholds. For the pigs that received a 25 Gy dose, progressive neurological impairment was observed starting at 2 months post-irradiation leading to the need for euthanasia by 3-4 months. On MRI, these two animals presented with diffuse white matter pathology consistent with the human disease that progressed to outright radiation necrosis and severe brain swelling. Histology was consistent with the final MRI evaluation. The pigs that received a 15 Gy dose appeared normal all the way to 6 months post-irradiation with no obvious neurological impairment or lesions on MRI or histopathology. CONCLUSION: Based on our results, a mini-pig model of radiation-induced brain injury is feasible though some optimization is still needed. The mini-pig model produced lesions on MRI that are consistent with the human disease and which are not seen in rodent models. Our data shows that the ideal radiation dose for this model likely lies between 15 and 25 Gy.

Vitamin E delta-tocotrienol and metabolite 13'-carboxychromanol inhibit colitis-associated colon tumorigenesis and modulate gut microbiota in mice.

The gut microbiota play important roles in colon cancer. Vitamin E δ-tocotrienol (δTE) and its metabolite δTE-13'-carboxychromanol (δTE-13') are known to have cancer-preventive effects, but their impact on gut flora during tumorigenesis and the role of the metabolite in δTE's beneficial effects remain to be determined. In the murine colitis-associated colon cancer (CAC) induced by azoxymethane (AOM) and dextran sulfate sodium (DSS), we show that δTE and δTE-13' inhibited the multiplicity of large adenomas (>2 mm2) by 34% (P<.05) and 55% (P<.01), respectively, compared to the control diet. δTE-13' diminished AOM/DSS-increased GM-CSF and MCP-1, and δTE decreased IL-1ß. Using 16S rRNA gene sequencing of fecal DNAs, we observe that δTE and δTE-13' modulated the composition but not the richness of gut microbes compared to the control. Both δTE and δTE-13' enhanced potentially beneficial Lactococcus and Bacteroides. The elevation of Lactococcus positively correlated with fecal concentrations of δTE-13' and its hydrogenated metabolite, suggesting that the metabolite may contribute to δTE's modulation of gut microbes. Furthermore, δTE-13' counteracted AOM/DSS-induced depletion of Roseburia that is known to be decreased in patients with inflammatory bowel diseases. δTE uniquely elevated (Eubacterium) coprostanoloigenes. Our study demonstrates that δTE and δTE-13' inhibited tumorigenesis, suppressed pro-inflammatory cytokines and modulated gut microbiota in a murine CAC model. These findings uncover new and distinct activities of δTE and δTE-13' and support the notion that the metabolite may play a role in δTE's anticancer and modulation of gut microbes.

Vitamin E alpha- and gamma-tocopherol mitigate colitis, protect intestinal barrier function and modulate the gut microbiota in mice.

Inflammatory bowel diseases (IBDs) including colitis are intestinal disorders characterized by chronic inflammation, barrier dysfunction and dysbiosis. Specific forms of vitamin E have been shown to attenuate colitis, but the mechanisms are not fully understood. The objective of this study is to examine the impact of α-tocopherol (αT) and γ-tocopherol-rich tocopherols (γTmT) on gut inflammation, barrier integrity and microbiota in dextran sulfate sodium (DSS)-induced colitis in mice. We observe that αT and γTmT mitigated DSS-caused fecal bleeding, diarrhea and elevation of IL-6. These vitamin E forms inhibited colitis-induced loss of the tight junction protein occludin, and attenuated colitis-caused elevation of LPS-binding protein in the plasma, a surrogate marker of intestinal barrier dysfunction, suggesting protection of gut barrier integrity. Consistently, αT and γT mitigated TNF-α/IFN-γ-induced impairment of trans-epithelial electrical resistance in human intestinal epithelial Caco-2 cell monolayer. Using 16S rRNA gene sequencing of fecal DNA, we observe that DSS reduced gut microbial evenness and separated microbial composition from healthy controls. In colitis-induced mice, γTmT but not αT separated gut microbial composition from controls, and attenuated DSS-caused depletion of Roseburia, which contains butyrate producing bacteria and is decreased in IBD patients. Canonical correspondence analysis also supports that γTmT favorably altered gut microbial community. In contrast, neither αT nor γTmT affected gut microbes in healthy animals. These results provide evidence supporting protective effects of αT and γT on intestinal barrier function and that γTmT caused favorable changes of the gut microbiota in colitis-induced mice.

Plasmid Acquisition Alters Vancomycin Susceptibility in Clostridioides difficile.

The increasing incidence of primary and recurring Clostridioides difficile infections (CDI), which evade current treatment strategies, reflects the changing biology of C difficile. Here, we describe a putative plasmid-mediated mechanism potentially driving decreased sensitivity of C difficile to vancomycin treatment. We identified a broad host range transferable plasmid in a C difficile strain associated with lack of adequate response to vancomycin treatment. The transfer of this plasmid to a vancomycin-susceptible C difficile isolate decreased its susceptibility to vancomycin in vitro and resulted in more severe disease in a humanized mouse model. Our findings suggest plasmid acquisition in the gastrointestinal tract to be a possible mechanism underlying vancomycin treatment failure in patients with CDI, but further work is needed to characterize the mechanism by which plasmid genes determine vancomycin susceptibility in C difficile.